Opposite Roles of RNase and Kinase Activities of Inositol-Requiring Enzyme 1 (IRE1) on HSV-1 Replication

Abstract

In response to the endoplasmic reticulum (ER) stress induced by herpes simplex virus type 1 (HSV-1) infection, host cells activate the unfolded protein response (UPR) to reduce the protein-folding burden in the ER. The regulation of UPR upon HSV-1 infection is complex, and the downstream effectors can be detrimental to viral replication. Therefore, HSV-1 copes with the UPR to create a beneficial environment for its replication. UPR has three branches, including protein kinase RNA (PKR)-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activated transcription factor 6 (ATF6). IRE1α is the most conserved branch of UPR which has both RNase and kinase activities. Previous studies have shown that IRE1α RNase activity was inactivated during HSV-1 infection. However, the effect of the two activities of IRE1α on HSV-1 replication remains unknown. Results in this study showed that IRE1α expression was up-regulated during HSV-1 infection. We found that in HEC-1-A cells, increasing RNase activity, or inhibiting kinase activity of IRE1α led to viral suppression, indicating that the kinase activity of IRE1α was beneficial, while the RNase activity was detrimental to viral replication. Further evidence showed that the kinase activity of IRE1α leads to the activation of the JNK (c-Jun N-terminal kinases) pathway, which enhances viral replication. Taken together, our evidence suggests that IRE1α is involved in HSV-1 replication, and its RNase and kinase activities play differential roles during viral infection.

Keywords: X-box binding protein 1 (XBP1); endoplasmic reticulum (ER); herpes simplex virus 1 (HSV-1); inositol-requiring enzyme 1 (IRE1); unfolded protein response (UPR).

Figure 1

Inositol-requiring enzyme 1 (IRE1α) expression is up-regulated by herpes simplex virus type 1 (HSV-1) infection. (A,B) HSV-1 infection increased IRE1α expression in HEC-1-A cells. HEC-1-A cells were infected with HSV-1 (strain HF) at multiplicity of infection (moi) = 1. (A) Total cellular protein was harvested at indicated time point p.i. (post infection) and subject to Western blot analysis using antibodies against total IRE1, gD-1 (Glycoprotein D) and GAPDH (glyceraldehyde 3-phosphate dehydrogenase). The number under each band represents the relative density of the band in comparison to the corresponding control normalized to GAPDH. Cells treated with Thapsigargin (Tg) (1 μM) harvested at 4 h was used as a positive control; (B) IRE1α transcription expression was determined by real-time PCR at various time points post-infection (p.i.). Total RNA was extracted, IRE1 mRNA level was measured by real-time PCR and normalized to GAPDH. Cells treated with Tg (1 μM) for 4 h was served as control. The data are presented as mean ± standard deviation (SD) from three independent determinations (* p < 0.05, ** p < 0.01); (C–E) Both IRE1α and gD expression were inhibited by PAA. HEC-1-A cells were pretreated or mock-pretreated with phosphonoacetic acid (PAA, 400 μg/mL) for 30 min and then infected or mock-infected with HSV-1 (HF) at moi = 1. Both protein and mRNA transcription levels of IRE1α and gD-1 at 24 h p.i. were determined. Columns and bars represent the mean ± SD of three independent experiments (** p < 0.01). All experiments were performed three times. Representative results are shown.

Figure 2

HSV-1 viral replication was inhibited by RNase activity of IRE1α. (A) The PCR approach for both unspliced X-box binding protein 1 (XBP1) and spliced XBP1 genes. Total RNA was extracted and reverse-transcribed, spliced XBP1 mRNA was amplified by real-time PCR using the specific primers [38]. The underlined nucleotides show the 26 bp intron sequence that is spliced out when ER stress occurred; (B) The double-stranded cDNA was synthesized by PCR using specific sense and anti-sense primers for both the unspliced and spliced XBP1 genes [38]; (C) The effect of viral infection on the spliced XBP1 (XBP1s) mRNA expression. HEC-1-A cells were infected with HSV-1 (HF) (moi = 1), total cellular RNA was extracted at the indicated times and the XBP1s mRNA expression was determined by real-time PCR (the upper part of the panel) and nucleic acid electrophoresis (the lower band) using the primers set as shown in the text. Cells were treated with Tg (1 μM) for 4 h served as control; (D) Spliced and unspliced XBP1 products of PCR amplification were showed by nucleic acid electrophoresis; (E,F) Consequence of Tg or STF-083010 treatment on HSV-1 replication. HEC-1-A cells were pretreated with serial concentrations of Tg or STF-083010 for 30 min and then infected with HSV-1 (HF) (moi = 1) for 24 h. The infectious viral particles from infected cells were titrated by measuring the plaque forming unit (PFU); (G,H) The effect of Tg or STF-083010 on HSV-1 gD expression. HEC-1-A cells were pretreated with serial concentrations of Tg or STF-083010 for 30 min and then infected with HSV-1 (HF) (moi = 1), gD-1 protein expression in the presence of drugs was determined via In-cell Western and normalized against DRAQ5 fluorescence at 24 h p.i.; (I) Effect of over-expression of XBP1s on HSV-1 replication. Vehicle pcDNA 3.1 or 3 ×FLAG-XBP1s plasmid was transfected into Hela cells for 48 h before cells were infected with HSV-1 (HF) (moi = 1). The cells were lysed after 24 h p.i. then XBP1s and gD-1 expressions were determined by Western blot analysis using antibodies against anti-FLAG and gD (the upper panel). Hela cells were infected with HSV-1/blue (moi = 1) after vehicle or 3 ×FLAG-XBP1s plasmid was transfected into cells for 48 h. The β-Gal activity was measured as described in the text 12 h p.i. (the lower panel); (J) The transcription levels of both EDEM and Herp were downregulated by HSV-1 infection. HEC-1-A cells were pretreated or mock-pretreated with Tg for 30 min then mock-infected or infected with HSV-1 (moi = 1) or treated with STF-083010 (60 μM). Total cellular RNA was extracted after 12 h p.i. and the mRNA levels were determined by real-time PCR. All experiments were performed three times. The representative results were shown. Data are mean values (±SD) of three independent experiments (* p < 0.05, ** p < 0.01), Error bars show standard deviations (SD) from three separate experiments.

Figure 2

HSV-1 viral replication was inhibited by RNase activity of IRE1α. (A) The PCR approach for both unspliced X-box binding protein 1 (XBP1) and spliced XBP1 genes. Total RNA was extracted and reverse-transcribed, spliced XBP1 mRNA was amplified by real-time PCR using the specific primers [38]. The underlined nucleotides show the 26 bp intron sequence that is spliced out when ER stress occurred; (B) The double-stranded cDNA was synthesized by PCR using specific sense and anti-sense primers for both the unspliced and spliced XBP1 genes [38]; (C) The effect of viral infection on the spliced XBP1 (XBP1s) mRNA expression. HEC-1-A cells were infected with HSV-1 (HF) (moi = 1), total cellular RNA was extracted at the indicated times and the XBP1s mRNA expression was determined by real-time PCR (the upper part of the panel) and nucleic acid electrophoresis (the lower band) using the primers set as shown in the text. Cells were treated with Tg (1 μM) for 4 h served as control; (D) Spliced and unspliced XBP1 products of PCR amplification were showed by nucleic acid electrophoresis; (E,F) Consequence of Tg or STF-083010 treatment on HSV-1 replication. HEC-1-A cells were pretreated with serial concentrations of Tg or STF-083010 for 30 min and then infected with HSV-1 (HF) (moi = 1) for 24 h. The infectious viral particles from infected cells were titrated by measuring the plaque forming unit (PFU); (G,H) The effect of Tg or STF-083010 on HSV-1 gD expression. HEC-1-A cells were pretreated with serial concentrations of Tg or STF-083010 for 30 min and then infected with HSV-1 (HF) (moi = 1), gD-1 protein expression in the presence of drugs was determined via In-cell Western and normalized against DRAQ5 fluorescence at 24 h p.i.; (I) Effect of over-expression of XBP1s on HSV-1 replication. Vehicle pcDNA 3.1 or 3 ×FLAG-XBP1s plasmid was transfected into Hela cells for 48 h before cells were infected with HSV-1 (HF) (moi = 1). The cells were lysed after 24 h p.i. then XBP1s and gD-1 expressions were determined by Western blot analysis using antibodies against anti-FLAG and gD (the upper panel). Hela cells were infected with HSV-1/blue (moi = 1) after vehicle or 3 ×FLAG-XBP1s plasmid was transfected into cells for 48 h. The β-Gal activity was measured as described in the text 12 h p.i. (the lower panel); (J) The transcription levels of both EDEM and Herp were downregulated by HSV-1 infection. HEC-1-A cells were pretreated or mock-pretreated with Tg for 30 min then mock-infected or infected with HSV-1 (moi = 1) or treated with STF-083010 (60 μM). Total cellular RNA was extracted after 12 h p.i. and the mRNA levels were determined by real-time PCR. All experiments were performed three times. The representative results were shown. Data are mean values (±SD) of three independent experiments (* p < 0.05, ** p < 0.01), Error bars show standard deviations (SD) from three separate experiments.

Figure 3

HSV-1 replication is regulated by kinase activity of IRE1α. (A) HSV-1 infection stimulated kinase activity of IRE1α in HEC-1-A cells. HEC-1-A cells were infected with HSV-1 (HF) (moi = 1). Cell lysates were prepared at the times indicated and subject to Western blot analysis using antibodies against total IRE1α, phosphorylated IRE1α, gD-1 and GAPDH; (B) XBP1 splicing induced by APY29 was attenuated by STF-083010 in a dose dependent manner. HEC-1-A cells were treated with both a serial concentration of STF-083010 and 3.125 μM of APY29, and then incubated for 24 h. Total RNA was prepared for measuring the expression of spliced and unspliced XBP1using reverse transcription-PCR analysis; (C,D) Viral replication was inhibited by APY29; (C) Confluent HEC-1-A cells cultured in a 96-well plate were pretreated with serial concentrations of APY29 and then infected with HSV-1 (HF) (moi = 1) for 24 h; (D) Before HEC-1-A cells were infected with HSV-1 (HF) (moi = 1), cells were incubated with both serial concentration of STF-083010 and 3.125 μM of APY29. gD-1 protein expression was determined via In-cell Western, and normalized by DRAQ5 fluorescence at 24 h p.i. Data are mean values ± SD of triplicate determinations (** p < 0.01). Representative results are shown; (E) Inhibition of IRE1α kinase activity had an inhibitory effect on HSV-1 replication in HEC-1-A cells. Cells were presence or absence of APY29 (3.125 μM) or STF-083010 (60 μM) or both of two drugs and then infected with HSV-1 (HF) at moi = 1. Total cellular cells lysates were prepared at 24 h p.i. and subject to Western blot analysis against antibodies to total IRE1α, phosphorylated IRE1α and gD-1; (F) Inactivation of IRE1α kinase activity suppressed HSV-1 replication in Hela cells. Hela cells were transfected with vehicle or IRE1 alpha KA-pcDNA3.EGFP or IRE1 alpha-pcDNA3.EGFP plasmid for 48 h and then infected or mock-infected with HSV-1 (moi = 1). The total and phosphorylated IRE1α, and gD-1 protein were determined by Western blot at 24 h p.i. All experiments were performed three times. The representative results were shown.

Figure 4

HSV-1 viral replication was regulated by kinase activity of IRE1α via JNK signal pathway. (A) HSV-1 infection induced the activation of JNK pathway in HEC-1-A cells. HEC-1-A cells were infected with HSV-1 (moi = 1). Whole-cell lysates were prepared from infected cells at the post infection times indicated and subject to Western blot analysis using antibodies against phosphorylated JNK, JNK, and gD. The number under each band represents the relative density of the band in comparison to the corresponding control normalized to GAPDH; (B) SP600125 had an inhibitory effect on viral replication in HEC-1-A cells. HEC-1-A cells were pretreated with serial concentrations of SP600125 for 30 min and then infected with HSV-1 (HF) (moi = 1). The total and phosphorylated JNK, and gD protein were analyzed by Western blot at 24 h p.i. The number under each band represents the relative density of the band in comparison to the corresponding control normalized to GAPDH; (C) IRE1α knock-down reduced HSV-1 viral gene replication in Hela cells. Hela cells were transfected with scramble siRNA as negative control (NC) or IRE1α siRNA for 48 h and then infected with HSV-1 (moi = 1), total RNA was extracted at 24 h p.i., both viral gD RNA level and IRE1α mRNA level were determined by real-time PCR and normalized to GAPDH. Data are means of three independent wells, and the bars represent the mean values ± SD, ** p < 0.01; (D) IRE1α knock down reduced the kinase activity of IRE1α and viral replication in Hela cells. Hela cells were treated according to material and methods Section 6, total cellular protein was harvested at 24 h p.i. and then phosphorylated IRE1α level, total IRE1α and gD expression were measured by Western blot using indicated antibodies; (E) IRE1α knock-down attenuated JNK activation stimulated by HSV-1 in Hela cells. Hela cells were treated according to material and methods Section 6, total cellular protein was harvested at 24 h p.i. preparing for Western blot analysis against gD, total and phosphorylated JNK antibodies; (F) HSV-1 stimulated JNK activation was suppressed by inactivation of IRE1α kinase activity in Hela cells. Hela cells were treated as describe in material and methods 7. Total cell lysates were prepared after 24 h p.i. for Western blot analysis using antibodies directed against gD, phosphorylated JNK and JNK; (G) IRE1α kinase activity inhibitor repressed JNK activation induced by viral infection in HEC-1-A cells. HEC-1-A cells were pretreated or mock-pretreated with APY29 (3.125 μM), only or both APY29 (3.125 μM), and STF-083010 (60 μM) for 30 min and then infected or mock-infected with HSV-1 (moi = 1). Total cell lysates were prepared and Western blot analysis was performed using specific antibodies at 24 h p.i. All experiments were performed three times. The representative results were shown.

Figure 4

HSV-1 viral replication was regulated by kinase activity of IRE1α via JNK signal pathway. (A) HSV-1 infection induced the activation of JNK pathway in HEC-1-A cells. HEC-1-A cells were infected with HSV-1 (moi = 1). Whole-cell lysates were prepared from infected cells at the post infection times indicated and subject to Western blot analysis using antibodies against phosphorylated JNK, JNK, and gD. The number under each band represents the relative density of the band in comparison to the corresponding control normalized to GAPDH; (B) SP600125 had an inhibitory effect on viral replication in HEC-1-A cells. HEC-1-A cells were pretreated with serial concentrations of SP600125 for 30 min and then infected with HSV-1 (HF) (moi = 1). The total and phosphorylated JNK, and gD protein were analyzed by Western blot at 24 h p.i. The number under each band represents the relative density of the band in comparison to the corresponding control normalized to GAPDH; (C) IRE1α knock-down reduced HSV-1 viral gene replication in Hela cells. Hela cells were transfected with scramble siRNA as negative control (NC) or IRE1α siRNA for 48 h and then infected with HSV-1 (moi = 1), total RNA was extracted at 24 h p.i., both viral gD RNA level and IRE1α mRNA level were determined by real-time PCR and normalized to GAPDH. Data are means of three independent wells, and the bars represent the mean values ± SD, ** p < 0.01; (D) IRE1α knock down reduced the kinase activity of IRE1α and viral replication in Hela cells. Hela cells were treated according to material and methods Section 6, total cellular protein was harvested at 24 h p.i. and then phosphorylated IRE1α level, total IRE1α and gD expression were measured by Western blot using indicated antibodies; (E) IRE1α knock-down attenuated JNK activation stimulated by HSV-1 in Hela cells. Hela cells were treated according to material and methods Section 6, total cellular protein was harvested at 24 h p.i. preparing for Western blot analysis against gD, total and phosphorylated JNK antibodies; (F) HSV-1 stimulated JNK activation was suppressed by inactivation of IRE1α kinase activity in Hela cells. Hela cells were treated as describe in material and methods 7. Total cell lysates were prepared after 24 h p.i. for Western blot analysis using antibodies directed against gD, phosphorylated JNK and JNK; (G) IRE1α kinase activity inhibitor repressed JNK activation induced by viral infection in HEC-1-A cells. HEC-1-A cells were pretreated or mock-pretreated with APY29 (3.125 μM), only or both APY29 (3.125 μM), and STF-083010 (60 μM) for 30 min and then infected or mock-infected with HSV-1 (moi = 1). Total cell lysates were prepared and Western blot analysis was performed using specific antibodies at 24 h p.i. All experiments were performed three times. The representative results were shown.